Macrophage colony stimulating factor plays an important role in many biological processes, including the development of blood cells and the regulation of the immune system. It also plays an important role in atherosclerosis, osteoporosis, and other diseases. Its role in atherosclerosis is the most significant because, if anything, this disease is the leading cause of death in most developed countries.
Atherosclerosis is an inflammatory process of the arteries that begins as a fatty deposit (primarily lipid-filled macrophages), progresses to a fibrofatty lesion (all the foregoing, plus a mesh of smooth muscle cells, T-cells, collagen, proteoglycans, and elastic fibers), and ultimately leads to a fibrous plaque (all the foregoing, plus dense connective tissue and necrotic debris). If a person fails to treat the plaque or modify the diet or other lifestyle characteristics that likely contributed to it, the lesion will eventually occlude most or all of the artery, and the person will experience symptoms of heart disease such as chest pain, may have a heart attack, and might even die. Even if he escapes a heart attack, he is still vulnerable to stroke, gangrene of the extremities, damage to internal organs, and other serious diseases. As a result, atherosclerosis is the single most important cause of death and disability in developed countries in America, Europe and Asia, and will soon overtake infection as the primary cause of death in the entire world (R. Ross., “The Biology of Atherosclerosis,” in E. Topol, Ed., Comprehensive Cardiovascular Medicine, Lippincott-Raven, Philadelphia (1998); C J L Murray, A D Lopez. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet. 349:1269–1276 (1997); T. A. Pearson, S. C. Smith, P. Poole-Wilson, “Cardiovascular specialty societies and the emerging global burden of cardiovascular disease: a call to action,” Circulation. 97:602–604 (1998).
Widely accepted scientific opinion maintains that atherosclerosis is a complex inflammatory response to injury of the blood vessel, and a crucial early event in atherosclerosis is injury of some form to the arterial endothelium. Oxidized low-density lipoprotein (Ox-LDL) is a major source of injury; toxins, viruses, homocystein, and mechanical injury are other sources. The interaction of these agents with the endothelium changes the endothelial cells, making them prone to further interaction with monocytes circulating in the blood. Injury to the endothelium permits monocytes and other inflammatory cell types to penetrate the outer layer of the endothelium, where they differentiate into macrophages. Macrophages proliferate within the developing lesion and load themselves with Ox-LDL and acquire a foamy appearance. The lipids activate the macrophages, which then express new genes for stimulating smooth muscle cells, producing enzymes, and stimulating other macrophages, all of which make a major contribution to the development of the atherosclerotic plaque. If injury to the endothelium continues, the smooth muscle cells migrate and proliferate into the subendothelial space and combine with connective tissue and other matter, thereby forming an atherosclerotic lesion. The developing lesion begins to occupy more and more volume, and eventually encroaches upon the inside of the blood vessel, thereby restricting the flow of blood, and frequently blocking the artery altogether.
The most effective known method to treat atherosclerosis is to modify, where possible, the risk factors associated with it (some risk factors, such as gender, cannot be modified). Proper exercise and diet, maintaining a healthy weight, drinking in moderation, and not smoking can significantly reduce atherosclerosis and the diseases (e.g., heart attack, stroke) it causes. This is easier said than done, of course: the prevalence of atherosclerosis suggests that most individuals have difficulty making the sacrifices that proper exercise and diet require or that they succumb to the disease nonetheless. The most common pharmacological treatment for atherosclerosis seeks to lower blood levels of LDL, the so-called “bad cholesterol.” This method of treatment is effective in reducing the complications of atherosclerosis in many individuals, yet it is not a cure: atherosclerosis remains a leading cause of death and disability. For these reasons, a basic medical text still teaches that “[t]reatment of established atherosclerosis is directed at its complications . . .” M. H. Beers and R. Berkow, eds., Merck Manual of Diagnosis and Therapy, 1658 (1999).
However, if it were possible to disrupt, at the cellular level, the process that causes atherosclerosis, one could direct treatment at the disease itself, instead of its complications. For example, if there were a method to inhibit the growth, survival or activation of macrophages (or of vascular smooth muscle cells that feature prominently in the diseased artery wall), one might be able to prevent the formation and progression of atherosclerosis, and this would obviously be far preferable to merely treating its complications. The present invention is directed to such a method.
Over the years, the inventor and his colleagues have accumulated evidence which strongly indicate that M-CSF/c-fms-dependent cell signaling contributes to both the development and the breakdown of atherosclerotic lesions by regulating the growth, survival and function of monocyte-macrophages and intimal smooth muscle cells. T. B. Rajavashisth et al., “Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified LDL,” Nature, 344:254–257 (1990); T. B. Rajavashisth et al., “Transcriptional activation of the macrophage-colony stimulating factor gene by minimally modified LDL: involvement of nuclear factor KB,” Arterioscler Thromb Vasc Biol., 15:1591–1598 (1995); T. B. Rajavashisth et al, “Transcriptional activation of the macrophage-colony stimulating factor gene by minimally modified LDL: involvement of nuclear factor kB,” Arterioscler Thromb Vasc Biol, 15:1591–1598 (1995); T. B. Rajavashisth et al., “Atherosclerosis: from risk factors to regulatory molecules,” in Encyclopedia of Human Biology, R. Delbacco and P. Abelson, eds. Academic Press, San Diego, Calif., 1:565–574 (1997). They have established this conclusion in vivo by studying atherogenesis in osteopetrotic (op) mice that lack M-CSF due to a point mutation in the M-CSF gene. Atherogenesis was induced either by feeding the mice a high fat, high cholesterol diet or by crossing op mice with either apolipoprotein (apo) E or LDL receptor (LDLR)-null mice to generate mice lacking both M-CSF and apo E or LDLR. J. H. Qiao et al., “Role of macrophage-colony stimulating factor in atherosclerosis: studies of osteopetrotic mice,” Am J Pathol, 150:1687–1699 (1997); T. B. Rajavashisth et al., “Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor deficient mice,” J Clin Invest, 101: 2702–2710 (1998). The absence of M-CSF in both apo E or LDLR-deficient mice results in decreased atherosclerosis despite marked hypercholesterolemia suggesting that inhibition of M-CSF function in the diseased vessel wall may confer antiatherogenic properties.
An early event in the development of atherosclerotic lesion also includes migration of SMC from the media to the intima of the artery wall, where they proliferate and load themselves with lipid and thus become foam cells. J. Thyberg et al., “Regulation of differentiated properties and proliferation of arterial smooth muscle cells,” Arteriosclerosis, 10:966–990 (1990); R. Ross, “The platelet-derived growth factor,” Cell, 46:155–169 (1986). Numerous observations suggest that during the atherogenic process SMC change their structure and function. ld. Although the precise mechanism of this phenotypic modulation remains unclear, several growth factors or cytokines appear to play an important role in this process. Intimal SMC derived from atherosclerotic lesions express increased levels of M-CSF isoforms and its receptor, c-fms. R. Ross et al., “Localization of PDGF-B protein in macrophages in all phases of atherogenesis,” Science, 248:1009–1012 (1990); R. N. Salomon et al., “Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma,” Proc Natl Acad Sci USA, 89:2814–2818 (1992). SMC can express c-fms when activated by cytokines such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), heparin-binding epidermal growth factor-like growth factor (HB-EGF), or phorbol esters. T. Inaba et al., “PDGF induces c-fms and scavenger receptor genes in vascular smooth muscle cells,” J Biol Chem., 267:17107–13112 (1992); T. Inaba et al., “Induction of sustained expression of proto-oncogene c-fms by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor, and its suppression by interferon-gamma,” J Clin Invest, 95:1133–1139 (1995); T. Inaba et al., “Synergistic effects of transforming growth factor β on the expression of c-fms, macrophage colony-stimulating factor receptor gene, in vascular smooth muscle cells,” FEBS Left, 399:207–210 (1996). These results imply that activated SMC may respond mitogenically to M-CSF, a possibility with important implications for atherogenesis; that is, since M-CSF expression is increased in atherosclerotic lesions compared to the normal artery, M-CSF may play an important role in mitogenically altering SMC, and cause them to contribute to the further development of atherosclerotic lesions.
To examine the role of M-CSF in proliferation of arterial SMC, the inventor has previously used cultured human aortic SMC (HASMC) as a primary cell model and the injured rat carotid artery as an in vivo model. T. Rajavashisth et al., “Role of induced expression of M-CSF and its receptor in the growth and proliferation of intimal smooth muscle cells,” Circulation, 88:1–468 (1993). His results indicate that induced expression of M-CSF and c-fms correlates strongly with the initiation of SMC proliferation observed in balloon-injured rat carotid artery. Intimal SMC express higher levels of M-CSF receptor as compared to medial SMC and proliferate in response to M-CSF, thus providing indirect evidence that induced M-CSF activity not only affects monocyte-macrophage proliferation but can also affect the proliferation of intimal SMC; this suggests a broader role for M-CSF in atherosclerosis. These findings are supported by observations in osteopetrotic (op) mice that totally lack M-CSF. In a periadventitial carotid arterial injury model in the op/op mouse, M-CSF deficiency markedly inhibits injury-induced intimal thickening and neointimal proliferation. Additionally, results from the effects of purified M-CSF on the growth of cultured HASMC favor the concept that intimal SMC acquire certain characteristics of monocyte-macrophages that may be related to their proliferation and phenotypic conversion into foam cells in atherosclerotic lesions. Recent studies from other investigators support the inventor's finding that M-CSF can modulate the growth of vascular SMC. T. Inaba et al., “Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: A mechanism for phenotypic change to phagocytic cells,” Mol Cell Biol, 16:2264–2273 (1996); T. Herembert et al., “Control of vascular smooth-muscle cell growth by macrophage-colony-stimulating factor,” Biochem J, 325:123–128 (1997).
M-CSF is a multifunctional protein that can stimulate the growth of monocyte-macrophages, trophoblasts, osteoclasts and vascular SMC and is necessary for the survival of these cells in culture and in vivo. E. R. Stanley et al., “CSF-1: a mononuclear phagocyte lineage specific hemopoetic growth factor,” J Cell Biochem, 21:151-159 (1983); R. J. Tushinski et al., “Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cell selectively destroy,” Cell, 28:71–77 (1982). Vessel wall bound M-CSF, in particular, can regulate recruitment, cell cycle, survival and proliferation of monocyte-macrophages leading to the genesis and progression of atheromatous lesions. The ability of M-CSF to stimulate the uptake and degradation of modified lipoproteins by upregulating scavenger receptors may lead to the removal of oxidized lipoproteins from the extracellular space and the generation of foam cells. S. Ishibashi, “Monocyte colony-stimulating factor enhances uptake and degradation of acetylated low-density lipoproteins and cholesterol esterification in human monocyte-derived macrophages,” J Biol Chem, 265:14109–14117 (1990); W. J. S. de Villiers et al., “Macrophage-colony stimulating factor selectively enhances macrophage scavenger receptor expression and function,” J Exp Med, 80:705–709 (1994); H. Shimano, “Human monocyte CSF enhances the clearance of lipoproteins containing apolipoprotein B-100 via both low-density lipoprotein receptor-dependent and -independent pathways in rabbits,” J Biol Chem, 265:12869–12875 (1990).
M-CSF exists in multiple isoforms. A single M-CSF gene gives rise to at least four distinct mRNAs due to alternative splicing in mice and humans. Rajavashisth et al., “Cloning and tissue-specific expression of mouse colony-stimulating factor mRNA,” Proc Natl Acad Sci USA, 84:1157–1161 (1987); D. P. Cerretti et al., “Human macrophage-CSF: alternative RNA and protein processing from a single gene,” Mol Immunol, 25:761–770 (1988). In mice, various tissues express a complex pattern of multiple M-CSF transcripts (ranging from 1.6 to 4.5 kb) in a highly tissue specific manner. These transcripts encode at least two distinct proteins, secreted as glycoprotein (sM-CSF) and proteoglycan (pgM-CSF) forms, and a third membrane bound (mM-CSF) isoform with cell-surface biological activity. M. B. Ladner et al., “Human CSF-1: Gene structure and alternative splicing of mRNA precursors,” EMBO J., 6:2693–2698 (1987); D. P. Cerretti et al., “Human macrophage-CSF: alternative RNA and protein processing from a single gene,” Mol Immunol, 25:761–770 (1988); E. S. Kawasaki et al., “Molecular cloning of a complementary DNA encoding human macrophage-specific colony stimulating factor (CSF-1),” Science, 230:291–296 (1985); S. Suzu et al., “Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species,” J Biol Chem, 267:4345–4348 (1992); C. W. Rettenmier et al., “Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and down modulation of CSF-1 receptors in NIH3T3 cells transformed by cotransfection of the human CSF-1 and c-fms,” Mol Cell Biol, 8:2378–2387 (1987). Precursors of mature M-CSF isoforms contain a common amino terminus, a spacer region of varying length, and a common trans-membrane domain at the carboxy terminus. The membrane bound precursor may be processed by different proteolytic activities located at different sites in the body, producing the secreted isoform of M-CSF. It is also possible that membrane bound M-CSF molecules function as cell-associated ligands stimulating biological response in a “juxtacrine” manner (i.e., requiring cell to cell contact). In this respect, the three different M-CSF isoforms would in effect extend the ligand binding domain of the amino terminus from the membrane, and thus allow a cell to stimulate a c-fms receptor on another cell located at a variable distance from the cell initiating the M-CSF signal.
Multiple isoforms of M-CSF utilize a single membrane bound receptor (c-fms), C. J. Sherr et al., “Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor,” Cold Spring Harbor Symp Quant Biol, 53:521–530 (1988), suggesting that this receptor may be a suitable target to inhibit M-CSF mediated cell signaling. This idea is directly supported by evidence indicating that systemic injection of a monoclonal antibody against murine c-fms in the LDLR-deficient mice results in markedly reduced atherosclerosis. T. B. Rajavashisth et al., “Monoclonal antibody against murine macrophage colony-stimulating factor receptor inhibits atherogenesis in LDL receptor-deficient mice,” Circulation 98:A1711,1198. These studies demonstrate that the monoclonal antibody specific to the ligand binding domain of the M-CSF receptor markedly inhibits atherosclerosis in LDLR-deficient mice. Furthermore, these studies strongly support the concept that M-CSF plays a key role in the genesis and progression of atherosclerosis. Although the exact mechanism by which anti-c-fms antibody confers its resistance to atherosclerosis remains to be elucidated, the observation that the disruption of M-CSF/c-fms signaling pathways inhibits atherosclerosis suggests that therapy based on disruption of M-CSF/c-fms signaling pathways could have potential as an effective antiatherogenic strategy.
Although anti-c-fms antibody inhibits atherosclerosis in mice, treating atherosclerosis (or any other disease in which inhibiting M-CSF would have a beneficial effect) by administering this antibody is not practical in humans. Producing such antibodies is expensive. It demands much laboratory time and many laboratory resources. To effectively administer it, one would need to deliver frequent intravenous injections to patients, which further limits its usefulness. There is therefore a need in the art for a method of inhibiting M-CSF, and, in particular, M-CSF/c-fms cell signaling, that does not depend on administering anti-c-fms antibodies; this arises from the even greater need for a method of treating atherosclerosis itself—the leading cause of death in much of the world—and not merely its complications. The inventor is the first to describe such a method, as set forth herein.